Cyclic peptides containing tryptophan and arginine as Src kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.03.124

A number of cyclic and linear peptides containing various combinations of amino acids were evaluated for their Src kinase inhibitory potency. Among all the peptides, cyclic decapeptide C[RW]₅ containing alternative arginine (R) and tryptophan (W) residues was found to be the most potent Src kinase inhibitor. C[RW]₅ showed higher inhibitory activity (IC ₅₀ = 2.8 μM) than C[KW]₅, L(KW)₅, C[RW] ₄, and C[RW]₃ with IC₅₀ values of 46.9, 69.1, 21.5, and 25.0 μM, respectively, as determined in a fluorescence intensity-based assay. Thus, the cyclic nature, the presence of arginine, ring size, and the number of amino acids in the structure of the peptide were found to be critical in Src kinase inhibitory potency. The IC₅₀ value of C[RW]₅ was found to be 0.8 μM in a radioactive assay using [γ-³²P]-ATP and polyE₄Y as the substrate. C[RW] ₅ was a noncompetitive Src kinase inhibitor, showing approximately fourfold more selectivity towards Src than Abl.

Full text of DOI:10.1016/j.bmcl.2013.03.124

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3230–3234
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Cyclic peptides containing tryptophan and arginine as Src kinase
inhibitors
Amir Nasrolahi Shirazi a, Rakesh Kumar Tiwari a, Alex Brown b, Dindyal Mandal a, Gongqin Sun b,
Keykavous Parang a,
⇑
a Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, RI 02881, United States
b Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI 02881, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of cyclic and linear peptides containing various combinations of amino acids were evaluated
for their Src kinase inhibitory potency. Among all the peptides, cyclic decapeptide C[RW]5 containing
alternative arginine (R) and tryptophan (W) residues was found to be the most potent Src kinase inhib-
Received 7 March 2013
Revised 23 March 2013
Accepted 27 March 2013
Available online 6 April 2013
itor. C[RW]5 showed higher inhibitory activity (IC50 = 2.8
lM) than C[KW]5, L(KW)5, C[RW]4, and C[RW]3
with IC50 values of 46.9, 69.1, 21.5, and 25.0 M, respectively, as determined in a fluorescence intensity-
l
based assay. Thus, the cyclic nature, the presence of arginine, ring size, and the number of amino acids in
Keywords:
Arginine
Cyclic peptides
Src
Tryptophan
Tyrosine kinase
the structure of the peptide were found to be critical in Src kinase inhibitory potency. The IC50 value of
C[RW]5 was found to be 0.8 lM in a radioactive assay using [c-
32P]-ATP and polyE4Y as the substrate.
C[RW]5 was a noncompetitive Src kinase inhibitor, showing approximately fourfold more selectivity
towards Src than Abl.
Ó 2013 Elsevier Ltd. All rights reserved.
Protein tyrosine kinases (PTKs) are enzymes that catalyze the
transfer of the -phosphate group from ATP to the hydroxyl group
molecule inhibitors targeting conserved ATP binding site of PTKs.
Furthermore, the diversity of amino acids in the peptide sequence
can be used to generate diversified structures.
c
in tyrosine residues in proteins. Src family kinases (SFKs) including
Src, Yes, Fyn, Fgr, Hck, Lck, Blk, Lyn, and Frk are nonreceptor tyro-
sine kinases that play critical roles in several cellular signaling
pathways. SFKs contain five distinct functional domains from the
N- to C-terminals, including fatty acid acylation, Src homology 3
(SH3), Src homology 2 (SH2), kinase (containing ATP and substrate
binding sites), and a C-terminal regulatory domain.1 Src contrib-
utes a significant role in cell proliferation, differentiation, and
motility in many normal cell lines.2 Overexpression of Src kinase
is observed in different types of cancer cells.3 Thus, the design of
potent Src kinase inhibitors has become a subject of major interest
in drug discovery.4,5 Most of the attention has been focused on
designing Src kinase inhibitors through blocking Src-dependent
substrate phosphorylation. The kinase domain inhibitors are de-
signed to inhibit the binding of ATP (ATP binding site inhibitors)6,7
or the protein substrate (substrate binding site inhibitors).8–10
Peptide-based inhibitors with enhanced specificity can be de-
signed and synthesized to target conserved regions of PTKs, such
as substrate binding site, SH3 domain, and the SH2 domain. The
sequence of amino acids in the structure of the peptide can mimic
the PTK substrates that target nonconserved regions of PTKs. Thus,
peptides can be designed to be more selective compared to small
In general, linear peptides adopt highly flexible conformations
in a solution that could lead to weak interactions with the desired
target. The optimized linear peptides usually show PTK inhibitory
potency in the micromolar range.11 Moreover, linear peptides have
limited stability and low bioavailability because of proteolytic
degradation by natural cellular protease. Thus, further structural
modification is required to enhance the stability, potency, and
selectivity of the linear peptides.
Cyclization strategy has been shown to be effective in
developing more potent and stable peptidic and peptidomimetic
agents.12–15 Cyclization of peptides has been commonly used to re-
duce the conformational freedom of the peptides. Introducing con-
formational constraints in peptides make them more stable
towards proteases. In addition, cyclization often results in higher
receptor binding affinity towards specific receptors possibly by
decreasing unfavorable entropic effects.16 For example, G7-
18NATE peptide (cyclo-[CH2CO-WFEGYDNTFPC]-amide) is a non-
phosphorylated tyrosine-containing peptide that was developed
to target specifically the SH2 domain of the growth factor receptor
bound protein 7 (Grb7).17 We have previously reported the synthe-
sis of a number of cyclic peptides, including analogues of Ac-CIY-
KYY and GpYEEI as Src kinase inhibitors and Src SH2 domain
binding ligands, respectively. Conformational constrained peptides
of CIYKYY significantly improved the inhibitory potency of the
⇑
Corresponding author.
E-mail address: kparang@uri.edu (K. Parang).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.124

A. Nasrolahi Shirazi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3230–3234
3231
H
N
NH2
NH2
NH3
H3N
NH
H
H
H
N
N
NH
O
N
NH
HN
O
H2N
HN
O
O
O
NH
O
NH3
O
NH3
NH
O
HN
HN
2HN
O
NH
O
NH
NH
NH
NH
HN
O
O
O
O
O
O
O
NH
HN
O
NH
HN
H3N
O
NH
O
O
O
NH3
O
H2N
HN
O
HN
N
NH2
HN
N
N
H
H
H
NH3
HN
HN
NH3
C[KF]4
NH2
H2N
C[KA]4
C[RW]4
H
NH2
NH2
N
OOC
OOC
NH
NH2
NH2
NH
H2N
HN
NH3
H
N
O
NH2
H
N
H
NH
N
O
HN
O
COO
HN
O
O
O
O
HN
O
NH
O
O
O
NH
NH
NH3
OOC
O
HN
COO
HN
O
COO
O
O
O
HN
O
NH
H3N
COO
O
OOC
NH
O
O
O
HN O
HN
O
HN
N
O
NH
OOC
NH
H2N
H
N
H
N
H
NH2
HN
NH
NH2
H2N
H3N
HN
OOC
NH2
H2N
C[RE]4
C[KE]4
C[RFEF]2
H2N
NH2
H2N
HN
HN
NH2
NH2
NH2
NH
HN
H2N
H2N
N
H
H
N
NH2
H
N
O
NH
NH
HN
N
HN
H2N
O
O
H
N
H
HN
O
O
NH
O
O
NH
O
NH
O
O
HN
HN
O
O
NH2
NH2
O
HN
NH
HN
O
NH
NH
NH2
NH
O
O
O
HN
O
HN
H2N
HN
O
O
O
H
N
O
NH
NH
N
O
HN
NH
N
NH2
N
H
NH
H
O
H
NH2
HN
N
H
H2N
NH2
NH
HN
NH
HN
H2N
H2N
HN
NH2
NH2
NH2
H2N
C[RW]5
C[RW]3
C[RF]4
Figure 1. Chemical structures of synthesized cyclic peptides.
peptides against active Src kinase when compared with the corre-
sponding linear peptide.18 Conformationally constrained analogs of
GpYEEI also showed significantly higher binding affinity towards
the Src SH2 domain.16
However, a detailed mechanistic study of the role of hydropho-
bicity, charge, and amino acid sequence on their Src inhibitory
potency of cyclic peptides remained elusive. In continuation of
our efforts to develop new cyclic peptide scaffolds as Src kinase
inhibitors,11,19 herein we report the design and evaluation of cyclic
peptides containing charged and hydrophobic residues for
potential applications as a new class of Src kinase inhibitors. The
hydrophobic residues such as aromatic side chains of phenylala-
nine and tryptophan were expected to occupy and/or interact with
a large hydrophobic pocket19 in the ATP binding site. Furthermore,
the indole ring of tryptophan could mimic the adenine base of ATP
in binding to the nucleotide binding site.
Seven cyclic octapeptides with various combination of L-amino
acids, C[RW]4, C[KF]4, C[KA]4, C[RFEF]2, C[KE]4, C[RE]4, and C[RF]4
(Fig. 1), were synthesized by employing 9-fluorenylmethyloxycar-
bonyl (Fmoc)-based chemistry according to our previously

3232
A. Nasrolahi Shirazi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3230–3234
O
NHBoc
NHBoc
NHBoc
NHBoc
NHBoc
NH2
O
(i) Fmoc peptide synthesis.
(ii) TFE/AcOH/DCM
O
O
H
O
H
O
O
H
N
NBoc
H
N
H
N
N
N
OH
H2N
N
H
N
H
N
H
N
H
O
O
O
O
O
H-Trp(Boc)-2-chlorotrityl
chloride resin
N
N
N
N
N
Boc
Boc
Boc
Boc
Boc
NH3
NH
(i) DIC,HOAT,DMF,DCM
(ii) TFA/anisole/thioanisole/EDT
H
H
N
N
O
H3N
O
NH
HN
O
O
NH
O
NH3
H
N
HN
NH
O
O
HN
NH
HN
O
O
O
N
H
N
H
HN
H3N
NH3
C[KW]5
Scheme 1. Solid-phase synthesis of [KW]5.
Table 1
Src kinase inhibitory activity of peptides
zation reaction. The dried crude linear protected peptide was dis-
solved in DMF/DCM (5:1 v/v). HOAt (4 equiv) and DIC (4.5 equiv)
were added to the mixture, and the solution was stirred for 4 h.
After the cyclization reaction completed the solvents were re-
moved under reduced pressure. Freshly prepared cleavage cocktail,
reagent R, TFA/thioanisole/EDT/anisole (90:5:3:2 v/v/v/v) was
added to the crude product. The mixture was stirred at room tem-
perature for 2 h. After precipitation of crude peptide in cold diethyl
ether (75 mL, Et2O), the crude peptide was lyophilized and purified
by reversed-phase Hitachi HPLC (L-2455) on a Phenomenex Prod-
a
Entry
Peptide
IC50 (lM)
1
2
3
4
5
6
7
8
Cyclic [KF]4
Cyclic [KA]4
Cyclic [RFEF]2
Cyclic [KE]4
Cyclic [RE]4
Cyclic [RF]4
Cyclic [RW]4
Cyclic [RW]3
Cyclic [RW]5
Cyclic [KW]5
Linear (RW)5
Linear (KW)5
Staurosporine
PP2
1567.6
P300
P300
P300
52.6
31.3
21.5
25.0
2.8
9
igy 10
l
M ODS reversed-phase column (2.1 cm ꢀ 25 cm) using a
10
11
12
13
14
46.9
7.1
69.1
0.6
gradient system to yield cyclic [KW]5. A similar procedure was em-
ployed for the synthesis of other cyclic peptides by using appropri-
ate resins and protected amino acids.
0.5
All L-cyclic peptides were evaluated for their Src kinase inhibi-
a
tory activity (Table 1). The effect of peptides on the activity of
Src kinase was assessed by TranscreenerÒ ADP2 Fluorescence
Intensity (FI) Assay. The results of Src kinase inhibitory activity
of cyclic peptides are shown in Table 1 (entries 1–7). Cyclic octa-
peptides [KF]4, [KA]4, [RFEF]2, and [KE]4 exhibited no Src kinase
The concentration of the compounds that inhibited the enzyme activity by 50%.
All the experiments were carried out in triplicate.
reported procedure.20 The design and selection of the cyclic pep-
tides was based on the presence of hydrophobic (e.g., W, F, A)
and charged residues (e.g., K, R, E).
As a representative example, the synthesis of cyclic [KW]5 is de-
picted in Scheme 1. The linear peptide (KWKWKWKWKW) was
assembled on the H-Trp(Boc)-2-chlorotrityl chloride resin. After
the final deprotection of the N-terminal Fmoc group by piperidine
in DMF (20% v/v), the side chain-protected peptide was cleaved
from trityl resin in the presence of the cleavage cocktail acetic
acid/TFE/DCM (1:2:7 v/v/v,) at room temperature to yield the side
chain-protected linear peptide that was used directly for the cycli-
inhibition at the highest concentration 300
and C[RE]4 exhibited modest inhibition of Src kinase with
IC50 = 21.5–52.6 M). Among these three peptides, C[RW]4 showed
the highest inhibition of Src kinase with an IC50 value of 21.5 M,
lM. C[RF]4, C[RW]4,
l
l
suggesting that tryptophan residues contributes in Src kinase
inhibition.
To study the effect of the ring size on the Src kinase inhibitory
activity, two other cyclic peptides namely C[RW]3 and C[RW]5
(Table 1, entries 8 and 9) containing six and ten alternative argi-
nine and tryptophan residues were synthesized, and their Src

A. Nasrolahi Shirazi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3230–3234
3233
H
H
N
H
N
H
N
H
N
N
O
O
O
O
O
H
N
H
N
H
N
H
N
H3N
OH
N
H
N
H
N
H
N
N
H
H
O
O
O
O
O
HN
H2N
HN
HN
HN
HN
NH2
H2N
NH2
H2N
NH2
H2N
NH2
H2N
NH2
L(RW)5
HN
O
HN
O
HN
O
HN
HN
O
O
H
H
N
H
N
H
N
N
OH
N
H3N
N
N
H
N
H
H
N
H
O
O
O
H
O
O
H3N
H3N
H3N
H3N
H3N
L(KW)5
Figure 2. Chemical structures of L(RW)5 and L(KW)5.
kinase inhibitory activities were compared with C[RW]4. C[RW]3
exhibited slightly lower Src kinase inhibitory activity (IC50 = 25.0
reactions varied in C[RW]5 concentration. Competition assays
varied ATP concentration while maintaining constant concentra-
tions of inhibitor, enzyme and substrate. Csk and Abl enzymes
were incubated with fixed and excess concentration of substrate
in a series of reactions in the presence of different concentrations
of C[RW]5 to calculate the turnover. The measured activity was
plotted versus the concentration of inhibitor to visualize graphi-
lM) than C[RW]4. On the other hand, decapeptide C[RW]5 showed
ninefold higher Src kinase inhibitory activity (IC50 = 2.8 M) versus
l
C[RW]4, suggesting that the ring size was a critical factor in
enhancing Src kinase inhibition in C[RW] series.
To investigate the role of the constrained cyclic nature of the
peptide, the corresponding linear (RW)5 (L(RW)5) (Fig. 2) was de-
signed and synthesized. C[RW]5 and L(RW)5 (Table 1, entries 9
and 11) share the same number and sequence of amino acids.
C[RW]5 and L(RW)5 represent constrained and flexible linear
peptides, respectively. L(RW)5 inhibited Src kinase with an IC50 va-
cally. The IC50 values of C[RW]5 were found to be 0.8
lM, 0.8 lM,
and 3.1 M for Src, Csk, and Abl kinases, respectively, in the
l
radioactive assay. These data suggested that the C[RW]5 is approx-
imately 3.9-fold more selective towards Src and Csk versus Abl.
To evaluate whether the cyclic peptide was competing with
ATP, further kinetic analysis was conducted. Csk and Src were incu-
bated with a range of concentrations of ATP while substrate turn-
over was recorded in the presence or absence of the inhibitor. The
plot showed that C[RW]5 followed a noncompetitive inhibition
pattern against ATP for both Csk and Src (Fig. 3). A double-recipro-
cal plot of the obtained data, followed by linear regression analysis
(r2 P 0.997) revealed that the Kcat/Km declined for both enzymes in
the presence of the peptide. For example, Kcat/Km was 1.91, 1.44,
lue of 7.1
(IC50 = 2.8
l
M, showing 2.5-fold lower inhibition than C[RW]5
l
M). These data suggests that the cyclic nature of the
peptide contributes to improving Src kinase inhibitory activity.
To get a better understanding of the effect of the arginine on the
Src kinase inhibitory activity of C[RW]5 and L(RW)5, two homo-
logues cyclic and linear peptides, C[KW]5 and L(KW)5 (Fig. 2) were
synthesized by replacing the arginine with lysine and their Src
kinase inhibitory activities were evaluated. C[KW]5 and L(KW)5
inhibited the Src kinase activity with IC50 values of 46.9
69.1 M (Table 1, entries 11 and 12), respectively, that are signifi-
cantly lower than the corresponding C[RW]5 (IC50 = 2.8 M) and
L(RW)5 (IC50 = 7.1 M). These data suggest that the presence of
arginine is required for optimal Src inhibitory activity.
lM and
and 1.34 minꢁ1 Mꢁ1 in the presence of 0, 0.5, and 1
l lM of the
l
inhibitor, respectively, suggesting that the catalytic efficiency of
Src was reduced in the presence of the peptide. The noncompeti-
tive inhibition pattern indicated that Src and Csk retained a signif-
icant capacity to bind ATP. These data suggests that C[RW]5 is
using other potential sites in the catalytic domain for generating
kinase inhibitory activity possibly by binding to an area of the ki-
nase domain with minimal overlap with the ATP binding site, such
as ATP binding site adjacent domains. More mechanistic studies
are required to determine the mode of inhibition and interaction
of positively charged and hydrophobic amino acids in C[RW]5 with
amino acids in the kinase domain.
l
l
In order to determine the selectivity of C[RW]5 toward Src
kinase, the IC50 values of the peptide were ascertained in two other
divergent nonreceptor protein tyrosine kinases, Abl and Csk in a
radioactive assay using [c-
32P]-ATP and polyE4Y as the substrate.
Enzyme activity and inhibition were characterized using the
following methods, adapted from previous work.14,15,21 Csk and
Src activity levels were measured from incubations with polyE4Y
(1 mg/mL). CrkL (0.5 mg/mL) was used as a substrate for Abl.
C[RW]5 was dissolved in 10% DMSO/90% kinase buffer solution
(KB10) and diluted in series as needed. IC50 measurement assay
To evaluate the cellular permeability of these two peptides, a
comparative intracellular uptake experiment was carried out by
using synthesized fluorescently labeled C[RW]5 and C[KW]5

3234
A. Nasrolahi Shirazi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3230–3234
than that of F0-C[WK]5 (Fig. 4). These results suggest that both
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
(a)
C-[RW]5 (1.0 µM)
C-[RW]5 (0.5 µM)
C-[RW]5 (0 µM)
C[RW]5 and C[KW]5 that contain similar number of amino acids
have efficient cellular permeability, but the presence of the argi-
nine in C[RW]5 improves the cellular uptake when compared to
the corresponding peptide C[KW]5 containing lysine.
In conclusion, a new class of cyclic peptides containing different
combinations of amino acids was evaluated for Src kinase inhibi-
tory activity. To the best of our knowledge, this is the first report
of cyclic peptides containing hydrophobic and positively charged
residues as Src kinase inhibitors. The combination of tryptophan
and arginine showed the highest potency to inhibit the Src kinase
activity compared to other peptides. The cyclic nature, ring size,
and the presence of arginine and tryptophan residues were found
to contribute significantly to the Src inhibitory activity. C[RW]5
with an IC50 value of 2.8 lM exhibited a significantly higher inhib-
itory potency compared to C[RW]4, L[RW]5, L(KW)5, and C[KW]4.
C[RW]5 was found to be selective to Src versus Abl kinase. The
peptide found to be a noncompetitive inhibitor to ATP. These data
suggest that C[RW]5 can be used as a scaffold for designing Src
kinase inhibitors with further structural modifications.
-0.01 0.00
-0.2
0.01
0.02
0.03
0.04
0.05
M -1
0.06
0.07
0.08
1/[ATP]
µ
0.04
(b)
C[RW]5 (0 µM)
C[RW]5 (0.5 µM)
C[RW]5 (1 µM)
Acknowledgments
0.035
0.03
We acknowledge the financial support from National Science
Foundation, Grant No CHE 0748555, and American Cancer Society
grant number RSG-07-290-01-CDD. We thank the National Insti-
tute of General Medical Sciences of the National Institutes of
Health under grant number 8 P20 GM103430-12 for sponsoring
the core facility.
0.025
0.02
0.015
0.01
Supplementary data
Supplementary data (cyclic and linear peptide synthetic proce-
dures and additional supporting data including mass spectra)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2013.03.124.
0.005
0
-0.02
-0.01
0
0.01
0.02
0.03
0.04
1/[ATP] µM-1
-0.005
References and notes
Figure 3. Pattern of inhibition of (a) Csk and (b) Src by C[RW]5; Lineweaver–Burk
plot of 1/V versus 1/ATP with varying concentration of C[RW]5 shows linear
noncompetitive inhibition.
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Figure 4. Cellular uptake of fluorescently-labeled peptides F0-C[KW]5 and F0-
C[RW]5 (5 M) after 1 h incubation.
l
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namely F0-C[RW]5 and F0-C[KW]5 and Fluorescence Activated Cell
Sorter (FACS). The labeled compounds were incubated with human
ovarian carcinoma (SK-OV-3) cells for 1 h at 37 °C. The cellular up-
take of F0-C[WR]5 was found to be approximately 1.8-fold higher
21. Kemble, D. J.; Wang, Y. H.; Sun, G. Biochem. 2006, 45, 14749.